The dynamic nature of the spliceosome stimulates our interest in understanding its role in gene expression. The long term objectives of the proposed research are to identify factors that are required for pre- mRNA splicing and to determine their role in splicing. The mechanism of pre-mRNA splicing will be studied in the fission yeast Schizosaccharomyces pombe. Yeast offer powerful classical and molecular genetic and biochemical approaches to the study of splicing. Fission yeast are a particularly informative model for studying universal splicing factors because they have many characteristics with regards to gene structure and splicing apparatus that are similar to metazoans. To initiate our studies, we have recently identified temperature sensitive fission yeast mutants that are defective in pre-mRNA splicing. The wild type genes defective in these mutants will be cloned by rescue of the temperature sensitive growth phenotype and sequenced. A combined genetic and biochemical approach will be taken to analyze the gene products and determine their function. Bacterially expressed gene products will be used in reconstitution studies and to raise antibodies that will be used to define functional regions of the nucleus and to aid in the identification of factors that interact with the original splicing gene products. This involves searching for multicopy number suppressors of dominant negative mutants. The collection of temperature-sensitive mutants will also be expanded and screened for mutants that accumulate pre-mRNAs and the U6 precursor. Since U6 itself is essential for splicing, the second assay has the potential for identifying novel U6- specific splicing factors that are possible regulatory elements. Through this unique combination of genetics, biochemistry and structural analyses the pre-mRNA splicing complex will be elucidated. Characterization of the splicing complex is the initial step in understanding the pre-mRNA splicing mechanism. The antibodies and mutants obtained from these studies will provide the foundation for future studies in which the conservation of the splicing mechanism may be investigated in detail.